Abstract: In order to acquire more accurate and complete intracellular metabolite information，and therefore to more realistically reflect the microbial intracellular metabolism，the systematic optimization of metabolomic sample preparation process was carried out from 3 aspects of quencher，extraction and solvent agent based on the GC-MS platform. The leakage of intracellular metabolites and cell injury with different quenchers was compared，and the results showed that 60% methanol/0.9% NaCl had the least leakage. The effects of extraction and solvent agent on relative abundance of metabolite distribution were investigated，and 60% methanol presented the significant extraction effect，and pyridine had the dissolving effect．The optimal sample pretreatment method for metabolomic analysis of Aureobasidium pullulans was determined as follows：pre-cooled 60% methanol/0.9% NaCl as quencher，ground in liquid nitrogen and repeated freezing and thawing for cell disruption，pre-cooled 60% methanol for the extraction of metabolites，and then the extracts were dissolved by pyridine and derivatized for 90 min under MSTFA at 40℃. By using this optimized protocol，more than 300 kinds of metabolic components were identified by GC-MS，including a large number of amino acids，organic acids and sugars. Thus our established method can be used to effectively analyze the A. pullulans metabolites.

Key words: metabolomics, Aureobasidium pullulans, GC-MS, pretreatment